PROJECT SUMMARY (Project 2; Lantz, Boitano, Runyan) Dusts in the arid Southwest of the United States, especially downwind of legacy mine tailings and smelters, such as the Iron King Mine and Humboldt Smelter Superfund site (IKMHSS), can contain high levels of arsenic and other contaminants. Inhalation of these dusts can lead to adverse health outcomes. Little data exist concerning the risk from exposure to arsenic containing dusts and the potential interactions between arsenic ingestion in water and dust exposures. Epithelial to mesenchymal transition (EMT) is vital for appropriate organ development, is also important in epithelial wound repair and plays a role in disease processes. Disruption of EMT during embryological development results in altered organ development while alterations in adults can result in dysfunctional wound healing that includes loss of epithelial barrier function and epithelial remodeling that increases risk for respiratory diseases. Overall Objective: To determine toxicological responses (alterations in EMT) to real world, arsenic containing dust inhalation and/or arsenic ingestion that exist around legacy mining sites. We will use dusts collected at the IKMHSS site and exposure levels that mimic those found at the site. Chemical and physical properties of arsenicals in the dusts (analyzed in Project 5) will be correlated with toxicity. Hypothesis: Dust borne arsenic exposure disrupts EMT both during development and in adults and this alteration in EMT results in airway dysfunction. Three Specific Aims will utilize in vitro and in vivo models to determine the effects of exposure to arsenic and arsenic containing dusts on EMT. Specific Aim 1) Use xCELLigence Real Time Cell Analysis (RTCA) screening to determine arsenic containing dust properties that cause cytotoxicity and to determine pathways involved in arsenic induced disruption of EMT. This will involve a high capacity test system that can screen large numbers of samples for toxicity. Screening will guide dust selection for inhalation studies and allow for correlation of dust properties with EMT changes and cellular toxicity. Specific Aim 2) Determine the effects of arsenic exposure on lung airway development. Alterations in lung function and anatomy in adult mice exposed during in utero and early postnatal development will be assessed and correlated with changes in EMT pathway gene expression. Specific Aim 3) Determine impact of arsenic on EMT in adult epithelium using primary cell cultures and in vivo measures. Adult mice will be exposed to arsenic where both in vivo and in vitro assessments using primary mouse conducting airway epithelial cells will be undertaken. Responses between developmental (Aim 2) and adult responses (this aim) will be compared to determine if similar pathways are involved in arsenic-induced EMT disruption for exposures occurring at these two distinct developmental times. Because real world dusts and concentrations will be used, these data will help to inform risk to humans from similar exposures. In addition, by combining in vitro and in vivo toxicity measurements with the chemical and physical characterization of arsenicals in the dust, we will be able to determine dust arsenical properties that predict toxicity.